Synthetic biology is an emerging field that aspires to design and build functioning biological circuits, including gene expression systems, using well-characterized biomolecular components and genetic modules (Atkinson et al., 2003; Basu et al., 2004; Becskei and Serrano, 2000; Blake et al., 2003; Elowitz and Leibler, 2000; Elowitz et al., 2002; Fung et al., 2005; Gardner et al., 2000; Guet et al, 2002; Guido et al., 2006; Hooshangi et al., 2005; tsaacs et al., 2003, 2004; satan et al., 2005; Kobayashi et al., 2004; Kramer et al., 2004, 2005; Kramer and Fussenegger, 2005; Malphettes and Fussenegger, 2006; Ozbudak et al., 2002; Pedraza and van Oudenaarden, 2005; Rosenfeld et al., 2002, 2005; You et al., 2004). Generating high-fidelity and inducible gene expression systems that can operate in an intact organism would assist experimental studies of cellular function, development, and disease.
Several techniques exist to regulate gene expression; however, each carries its own caveat in function. It was discovered that both the tetR and lacI Escherichia coli repressor systems function in mammalian cell tissue culture and in mice (Brown et al., 1987; Gossen and Bujard, 1992; Hu and Davidson, 1987; Scrable, 2002), which proved to be a great advance in the understanding of cellular function. Other similar techniques have been used, including the inducible Gal4/UAS system, to control gene expression at the transcriptional level (Ornitz et al., 1991). While these techniques offer good repression, they exhibit leakiness that precludes the gene of interest from being completely turned off. Studies performed with double-stranded RNA in the nematode Caenorhabditis elegans revealed a sequence-specific RNA-mediated pathway for turning off gene expression (Fire et al., 1998; Guo and Kemphues, 1995). This process, known as RNA interference (RNAi), has been adapted for use in tissue culture and mammals with the introduction of small interfering RNAs (siRNAs) and short-hairpin RNAs (shRNAs). RNAi has revolutionized biological research; however, targeting locations on mRNAs for robust knock-down is empirical and often requires screening very large numbers of selected mRNA sequences (Paddison et al., 2004). Additionally, off-target effects can affect genes not related to the gene of interest. A commonly used method to activate or inactivate gene expression in mice involves the use of site-specific cre recombinase (cre). cre, which was derived from bacteriophage P1, mediates the deletion of a DNA sequence flanked by a pair of cre recognition sequences, called IoxP sites (Sternberg and Hamilton, 1981). A disadvantage of this approach is that it is dependent on the coexpression of the transgene cre, which causes a permanent genetic event, restricting any regulation of gene expression. This approach can be made inducible with the application of cre-ERTM or ERT2 fusion proteins; however, the inducing ligand, tamoxifen, can be toxic at the dosage levels required for recombination (Danielian et al., 1998; Imai et al., 2001). The caveats associated with these systems for regulating gene expression make it difficult to study many fundamental questions concerning cellular processes and disease.
Further, one of the major obstacles in various field of biotechnology (including but not limited to gene therapy and plant biotechnology) is the difficulty to achieve cell or tissue specificity. Transcription is an essential process for every living organism to convert abstract genetic information into physical reality.
The promoter is a major component to drive transcription. Some promoters are active in every tissue termed constitutive promoters, such as for e.g. actin promoters, while other promoters are only active in limited tissues. It is quite often that a given promoter is predominantly active in one tissue type but weakly expressed in some other tissues and allows some level of basal or background expression and are termed “leaky promoters” herein. Use of such leaky promoters is undesirable for agriculture and pharmaceutical application because the unintended expression of gene-of-interest resulted from leaky promoters and risk of detrimental effects to crops or patients. It certainty would not meet requirement of regulatory agency.
Unfortunately, for most of the present expression systems the expression of the introduced transgene is not limited to the target site, such as a tumor site, as the available promoters such as tissue-specific promoters or tumor-specific promoters are leaky and therefore the transgene is expressed in other tissue and thereby limits the use and efficiency of such approaches. While tissue specific promoters may add a further degree of control of transgene expression and selectivity, few of these have been validated in vivo and all have some degree of non-specific activation or “leakiness”. A versatile mechanism for controllable gene expression is therefore highly desired for gene therapy. A mechanism for controlling gene expression should ideally include both spatial and temporal control of gene expression.
For example a major problem in chemotherapy and radiation therapy for cancer is the difficulty in achieving tumor-specific cell killing. The inability of radiation or cytotoxic chemotherapeutic agents to distinguish between tumor cells and normal cells necessarily limits the dosage that can be applied. As a result, disease relapse due to residual surviving tumor cells is frequently observed, and thus there exists a clear need for alternative non-surgical strategies. Development of gene therapy techniques is approaching clinical realization for the treatment of neoplastic and metabolic diseases, and numerous genes displaying anti-tumor activity have been identified. However, the usefulness of gene therapy methods has been limited due to systemic toxicity of antitumor polypeptides encoded by gene therapy constructs (Spriggs & Yates (1992) in Bentler, ed., Tumor Necrosis Factor: The Molecules and Their Emerging Roles in Medicine, pp. 383-406 Raven Press, New York, N.Y.; Sigel & Puri (1991) J Clin Oncol 9:694-704; Ryffel (1997) Immunopathol 83:18-20). Problems with current state-of-the art gene therapy strategies include the inability to limit gene expression of the therapeutic gene specifically in the target cells. Non-specific gene expression of the therapeutic gene can lead to toxicity and undesired phenotypes in cells that are not the intended targets.
For example, manipulation of the p53 gene suppresses the growth of both tumor cells and normal cells, and intravenous administration of tumor necrosis factor alpha (TNFα) induces systemic toxicity with such clinical manifestations as fever and hypertension. While attempts have been made to overcome these problems, by using strategies utilizing tissue-specific promoters to limit gene expression to specific tissues and the use of heat (Voellmy R., et al., Proc. Natl. Acad. Sci. USA, 82:4949-4953 (1985)) or ionizing radiation inducible enhancers and promoters (Trainman, R. H., et al., Cell 46: 567-574 (1986); Prowess, R., et al., Proc. Natl. Acad. Sci. USA 85, 7206-7210 (1988)) to enhance expression of the therapeutic gene in a temporally and spatially controlled manner, these approaches have limited applicability die to residual background expression and inaccessibility of the heat of ionizing radiation to reach the target tissue.
Furthermore, controllable, tissue specific expression of transgenes is a primary goal in transgenic animals that serve as experimental models of disease. Early attempts at controlling transgene expression in transgenic mice have often employed transgenes that had been operably linked to tetracycline-responsive promoters or to rapamycin-responsive promoters.
The first tetracycline-controlled promoter was constructed by fusing the operator sequence of the E. coli tetracycline-resistance gene (tetO) to the minimal promoter sequence of the human cytomegalovirus immediate-early gene (hCMV IE), (Gossen, M. and Bujard, H., Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. NatL Acad Sci. USA 89(12), 5547-5551, 1992.) A promoter such as a tetO/hCMV is activated when it binds to a fusion protein constructed from the tetracycline-controlled transactivator (tTA), which contains a tetO binding protein tetracycline repressor (tetR), plus a transcription activator, virion protein 16 (VP 16), from herpes simplex virus. In the absence of tetracycline, this fusion protein binds to the tetO/hCMV promoter and activates transcription of the exogene. In the presence of tetracycline, exogene transcription is blocked because tetracycline binds to the transactivator (tTA) and interferes with its binding to the tetO/hCMV promoter. Since tetracycline down-regulates transgene expression, this is called the “tet-Off” promoter system. However, the tet-Off promoter system is associated with leaky gene expression which complicates the use of this system for basic research or pharmaceutical application. Furth and colleagues first used the tet-Off tetracycline-controlled promoter in transgenic mice that expressed the reporter transgene, luciferase, under control of the tetO/hCMV promoter. When tetracycline was absent, luciferase expression was observed in numerous tissues. When tetracycline was provided subcutaneously, the luciferase activity was significantly reduced to low, but still detectable, background levels, thus inducing “leakiness” of the tet-off promoter.
This leaky transgene expression was observed with two different plasmid delivery systems, the paired plasmid system of Furth (Furth, P. A., St. Onge, L., Boger, H., Gruss, P., Gossen, M., Kistner, A., Bujard, H., and Henninghausen, L., Temporal control of gene expression in transgenic mice by tetracycline responsive promoter. Proc. Natl. Acad Sci. USA 91(20), 9302-9306, 199) and the combined system of Nathalis (Schultze, N., Burki, Y., Lang, Y., Certa, U., and Bluethmann, H., Efficient control of gene expression by single step integration of the tetracycline system in transgenic mice. Nature Biotechnology 14(4), 499-503, 1996.).
Similarly, leaky transgene expression has also been observed in transgenic models that utilized the tet-Off system linked to a tissue specific promoter, the cardiac-specific, α-myosin heavy chain promoter (α-mhc) (Yu, Z., Redfern, C. S., and Fishman, G. I., Conditional transgene expression in the heart. Circ. Res. 79(4), 691-697, 1998). In these studies, the leaky transgene expression was observed in various tissues, such as kidney, skeletal muscle, pancreas, and live. Even stronger leaky gene expression was observed in cardiac tissues.
The tetO/hCMV genetic system has also been modified to allow tetracycline to induce, rather then inhibit, transgene expression. Such a modified system employs the reverse tetracycline-controlled transactivator (rtTA), comprised of a mutated tetO binding protein, rtetR (tetracycline repressor, rtTA) linked to VP16 (Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., and Bujard, H., Transcriptional activation by tetracyclines in mammalian cells. Science 268(5218), 1766-1769, 1995). When tetracycline is absent, the rtTA cannot bind to the tetO in the tetracycline-controlled promoter. When tetracycline is present, it binds to the rtTA which allows the rtTA to bind to the tetO in the promoter and up-regulate transcription of the exogene. Since tetracycline induces gene expression, this is called the “tet-On” promoter system.
While leaky gene expression is less prevalent in transgenic mice in which the transgene is under control of the tet-On promoter as compared to the mice in which the transgene is under control of the tet-Off promoter. However, use of the tet-On promoter is not able to eliminate residual leaky gene expression. In addition, transgenic mice whose transgenes are responsive to rapamycin have also been shown to express detectable background levels of transgene transcription in the absence of rapamycin. Such “leaky” transgene transcription seriously compromises studies with these transgenic mice.
Accordingly, it is desirable to have new methods and systems for producing transgenic animals, particularly transgenic mice, which have little to no background transgene transcription, particularly in specific tissues.
In a similar approach to the tet system, ionizing radiation has been used to activate a radiosensitive promoter, e.g. the EGR-1 promoter (Weischelbaum et al. (1994) Cancer Res 54:4266-4269; Hallahan et al. (1995) Nat Med 1(8):786-791; Joki et al. (1995) Hum Gen Ther 6:1507-1513). Another alternative design relies on endogenous control of gene expression. For example, the CEA promoter is selectively expressed in cancer cells (Hauck & Stanners (1995) J Biol Chem 270:3602; Richards et al. (1995) Human Gene Ther 6:881-893).
In the past, several approaches have been attempted to solve leakiness problem in plant gene expression without much success. By conducting a series of deletion of promoter sequence, one might eliminate the sequence in the promoter region which contributes to the leaky expression. For example, a deleted version of TbRB7 promoter drives GUS reporter gene expression in nematode feeding cells in the root upon nematode infection. Leaky expression, however, in flower tissue is still unsolved (Opperman C H et al., Science 263:221-223, (1994)). By making a chimeric promoter, i.e. a minimal promoter (e.g. 35S promoter) plus tissues-specific regulatory elements, one might restrict gene expression in desired tissues. However, if tissue-specific regulatory elements are leaky, the chimeric promoter will be leaky as well.
Several other methods to control leakiness of transgene expression have been explored, although each has their limitations. For example, U.S. patent application No: 2003/0045495 disclosed a modified inducible systems for selective expression of therapeutic genes by hyperthermia, although its utility is limited as hypothermia is also difficult to be applied to discrete cells or small tissue areas. Similarly, U.S. patent application No: 2001/0049828 discloses a method and system for controlling the expression of transgene products in specific tissues in a transgenic animal, which is based on an interaction of various transactivators in a transgenic mouse system. The transgene expression is controlled by antisense which is under control of the transactivator and tissue-specific promoters, thereby suppressing expression in certain tissues. However, this system is severely limited by its requirement of the antisense gene to be targeted to an endogenous gene sequence, for example the antisense targets the transgene being expressed and this could lead to non-specific effects if the antisense target gene is expressed endogenously in the cell, or in circumstances where the target gene is a member of a gene family also expressed in the cell. Accordingly, the use of this system is limited by being highly complicated and relying on several expression constructs and transgenic transcription factors. Similarly, U.S. patent application No:2002/0065243 discloses a system whereby an antisense to a tet repressor is expressed under the control of a constitutive promoter. Another system as disclosed in U.S. patent application No: US2002/0022018 describes tissue-specific deletion or destruction of the expression by tissue-specific expression of a Cre recombinase. As a result of Cre recombinase expression, the transgene floxed by LoxP sites in that tissue will be cut by the action of the Cre recombinase. Consequently, unwanted transgene as well as viral gene expression are prevented. However, due to leakiness of the promoter driving Cre expression, expression is expected to be lowered also in the target tissue itself, thereby decreasing overall efficiency of this approach.
Gene transfer involves the transfer of foreign genetic material into a cell such that the foreign material or transgene is expressed. This process is used in applications such as, for example: gene therapy, production of recombinant biologicals, genetic diagnosis, and drug screening. But despite recent reports of success in the most challenging of these fields, in vivo gene therapy of human diseases (Kay et al., 2000; Cavazzano-Calvo et al., 2000), the construction of new expression vectors has occupied the attention of the many workers eager to achieve high levels of gene expression in a regulated manner (reviewed in Agha-Mohammadi and Lotze, 2000).
In most cases, the ultimate goal of gene transfer is to introduce an expression vector that provides for production of a gene product for a period sufficient for a therapeutic or prophylactic effect, which period may be relatively short (e.g., a few hours to a few days) or may be for long periods (e.g., several weeks to one or more years). One important aspect of gene-based therapy could involve regulating expression in such a manner that gene expression is restricted spatially and temporally to cells or tissues that are affected by a disease. Such regulation requires that the gene be delivered to the target cell or tissue in a substantially latent state, so that it does not change or significantly affect the phenotype of the target in the absence of disease. Where and when the disease is active, it would be desirable that the latent gene should then be induced (e.g., spatially, temporally, or both) in a manner that will counteract disease symptoms and, conversely, ceases expression as the disease symptoms subside. To simplify, this requires that the gene be regulated by a tight on/off switch that can respond to a stimuli to switch the expression of the gene on/off.
A critical feature of such regulated gene expression is called the silencer-inducer ratio: expression of the transgene measured under inducing conditions divided by the amount of expression without induction (i.e., basal expression). Basal expression is a level of gene expression under non-inducing conditions and is a measure of the promoters “leakiness”. This ratio should be high (e.g., at least about 25- to 1,000-fold) and sufficiently regulatable by appropriate control of inducing conditions. Another critical feature is substantially silenced (or repressed) gene expression in the non-induced state.
This requirement for a tight on-off switch in regulating expression of a transgene is widely acknowledged and the absence of such regulation is considered to be one of the major limitations for many gene transfer applications. Regulated expression of transgenes, both positive and negative, has been described in prokaryotes (e.g., the Lac operon) and in mammals (e.g., Tetrepressor and activator, progesterone or ecdysone receptor) (reviewed in Agha-Mohammadi and Lotze, 2000). Each of these systems involves binding of an extrinsic modulator to a protein involved in transcription: tetracycline or doxycycline in the Tet regulatory system; RU486 or rapamycin in the progesterone and FKBP regulatory systems, respectively. The latter two systems require multiple vectors to deliver the target gene and the different regulatory components. In all of these systems, allosteric changes determine the DNA binding affinities of positive- and negative-acting transcriptional factors and thereby control an on-off switch (Freundlieb et al., 1999). However, these systems are all limited due to levels of “leakiness” of the promoters. 1251 Accordingly, there remains substantial need for improvement of methods and systems for inducing gene expression in a highly controllable manner, while maintaining control of promoter leakiness to ensure no background transgene expression, particularly for therapeutic uses and gene expression in specific tissues.